ABSTRACT The hallmark of systemic lupus erythematosus (SLE) is the production of antibodies to nuclear antigens such as ribonucleoproteins and DNA, with the resulting immune complexes causing systemic immune activation and tissue inflammation. High-affinity IgG antibodies to double-stranded DNA (dsDNA) are particularly pathogenic and associate with the severity of tissue damage. The mechanisms of tolerance to self-DNA and of its breakdown in SLE are poorly understood. We have studied these mechanisms by focusing on DNASE1L3, a secreted DNase that is mutated in several cases of early-onset familial SLE. We found that DNASE1L3-deficient mice develop a massive anti- dsDNA antibody response, whereas the response to other antigens was absent or delayed. This response and the ensuing immune activation and tissue damage required innate immune signaling through the adaptor protein MyD88. DNASE1L3-deficient mice and human patients showed the accumulation of genomic DNA within circulating apoptotic microparticles, and this DNA was recognized by autoantibodies in a DNASE1L3-sensitive manner. Thus, DNASE1L3 maintains tolerance to self-DNA by digesting potentially antigenic cell-extrinsic DNA in apoptotic microparticles. The proposed work will apply the newly developed conceptual framework and experimental tools to analyze the fundamental mechanisms of anti-DNA immune responses and their relevance to human SLE. In Aim 1, we will use DNASE1L3-deficient mice as a model of primary anti-dsDNA reactivity to characterize the nature and regulation of DNA-reactive B cells. In Aim 2, we will characterize innate immune mechanisms of anti-DNA antibody response, particularly the identity of MyD88-dependent sensing pathways. In Aim 3, we will translate our findings to human SLE patients, studying the antibody response to DNASE1L3-sensitive chromatin on microparticles. Collectively, these studies would provide insights into the origin and mechanisms of the pathogenic anti-DNA responses in SLE, and facilitate targeted approaches towards their therapeutic blockade.